IgM has been shown to direct systemic antibody response through the use of complement activation, especially in response to larger antagonists such as viruses or bacteria. This increased complement activation also leads to substantially higher complement-dependent cytotoxicity (CDC), making IgMs more effective than IgGs in killing target cells without requiring any immune cells.
As expected, Protein A agarose resin was capable of binding human IgG2, but not IgG3, whereas both LigaTrap-G and Protein G agarose resin were capable of binding both target antibodies. Both LigaTrap-G and Protein A resins afforded remarkable values of product yield and purity.
while initially captured by the LigaTrap-G resin, the serum albumin was removed in the washing step. Unlike what observed with CHO fluids, the binding of albumin from HEK-derived feed samples is rather conspicuous. This can be attributed to the much lower ratio of IgG vs. BSA in the HEK fluids compared to that of CHO fluids.
Resin equilibration prior to load and post-load washing were both performed with a buffer at high ionic strength (0.35 M NaCl in PBS at pH 7.4), to prevent or disrupt non-specific binding of serum albumin by electrostatic interaction. The elution of an IgG product whose purity (94.6%) is on par with the values obtained with commercial Protein A (96.7%) and Protein G (96.9%) resins. LigaTrap-G afforded a product yield (62.9%) comparable with that given by Protein A agarose resin (68.1%), and both were decidedly higher than that obtained with Protein G agarose (40.6%).
The LigaTrap-M resin outperformed the CaptureSelectTM IgM resin in terms of both product purity (93.1% vs. 76.2%) and yield. Poor binding selectivity by the 2-mercaptopyridine, in fact, resulted in a conspicuous amount of albumin eluted in 1 M ammonium sulfate.
The IgM yield of 75.1%, as measured by IgG-specific ELISA, indicates that, despite the low concentration of IgM in the feed (0.1 mg/ mL, 0.13 μM, corresponding to 1 – 3% of the total protein content in the feed), the peptoid ligand was successful in recovering the product.
The LigaTrap-M resin was tested with an IgG-depleted human serum of the IgA and IgM contents. The electrophoretic analysis of the concentrated eluate indicates that IgA and IgM are both present and at high purity (91.7%). Subsequent SEC fractionation returned separate IgM-enriched (lanes 1–6) and IgA-enriched (lanes 18–24).
